Measuring molecular binding or interactions is a basic task for understanding many biological processes and for developing relevant applications. A particularly important application is to determine the binding properties of drugs with their corresponding membrane protein receptors, which are the largest type of drug targets.1 Molecular binding is quantified by kinetic constants, which have, thus, become a criterion in preclinical drug screening.2,3 
Advances in structural biology have led to an exponential growth in the number of membrane proteins with determined 3D structures. However, as noted above, in order to understand the cellular functions of membrane proteins, it is also necessary to determine the interaction kinetics of the membrane proteins with various molecules. This is because cells perform many functions, including communication, via the interactions of their membrane proteins with molecules in the extracellular medium. A capability to quantify membrane protein interactions with molecules is also critical for discovering and validating drugs because most drug targets are membrane proteins. Despite the importance, developing such a capability that can measure the interactions of molecules with membrane proteins in the natural lipid environment has been a difficult task.
One traditional method for determining the kinetic constants is to extract molecular receptors from cells, immobilize them on a solid surface after purification, and then expose them to the drug for binding.4 Although useful, such methods can be problematic, especially when the receptors are membrane proteins,5 which currently count for more than a half of the drug targets.6 Due to their unique amphiphilic structures, it is difficult to ensure that the purified membrane proteins retain their native structures and functions.4 Because of the heterogeneous nature of cells, it is also important to study each of the individual cells. These capabilities, if developed, will benefit not only drug discovery, but also drug resistance study, which is a common but difficult problem in medicine.7-9 
Typically, methods for studying molecular interactions use radioactive or fluorescent labels. These end point assays do not provide kinetic constants that are needed to quantify the membrane interactions and functions. To determine the kinetic information, the current practice involves extracting membrane proteins from cells, purifying them from the extracts, immobilizing the purified proteins on a solid surface, and then exposing them to a ligand for kinetic study. The procedures are not only laborious, but also prone to alteration of the native functions of membrane proteins, especially integral membrane proteins that are permanently attached to the membrane. Furthermore, the isolation of membrane proteins from their native cellular environment prevents one from studying the allosteric effect in the molecular interactions, and examining heterogeneous nature of cells. A more serious limitation of the existing technologies is that the detection signal diminishes with the mass of the molecule, making them difficult for detecting small molecules, which play many important roles in cellular functions, and represent the vast majority of the existing drugs.
Various methods have been developed for in situ measurement of drug-receptor binding. A popular method is kinetic exclusion assay, which measures the concentration of free drugs remaining in the supernatant after the binding equilibrium in cell suspension is achieved with a labeled detection technology.15-17 This is an end-point assay, and not suitable for extracting the kinetic constants, including the association and dissociation constants. Furthermore, the use of labels is not only labor intensive but may also affect the native binding behaviors of the molecular receptors. Label-free technologies, such as quartz crystal microbalance, have been developed for studying drug binding properties.18 Although useful, they lack spatial resolution required to study the variability between different individual cells, map heterogeneous distribution of receptors in the cell membrane, and distinguish non-specific binding onto the sensor surface from specific binding to the receptors on the cells. Another label-free detection technology is surface plasmon resonance (SPR) technique that can monitor the lectinglycoprotein interactions in single cells.19 However, like the quartz crystal microbalance, SPR signal is proportional to the mass of the molecule (e.g., drug), which has limited sensitivity for detecting drug molecules with typically small molecular masses.
The present invention overcomes the shortcomings discussed above and, for the first time, discloses a system that can detect the binding of both large and small molecules with the molecular receptors in single cells, and analyze the corresponding binding kinetic constants. The method can be applied to measure the binding of drugs with their membrane receptor targets in the native cellular membranes, and to analyze cell-to-cell variability of the binding kinetics by measuring mechanical deformation of cells upon interactions of the cellular membrane proteins with molecules in the extracellular medium. A capability of real time analysis of the interactions in single cells by analyzing the mechanical deformation with sub-nm resolution is also provided for the first time. For small molecules, the present method represents the first kinetic measurement while the equilibrium constants extracted from the present method are consistent with those obtained with endpoint radioactive labeling assay. The imaging capability allows revelation of cell-to-cell variability of difference cells, and region-to-region variability within the same cell. The detection principle of the present invention may also be used to monitor the electrical activities in neurons.